Dihexyl fumarate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 7th of July 2004. Experimental Completion Date: 19th of July 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

pre-incubation assay performed in 50 µL of test solution instead of 100µL. Reason: In the pre-incubation test higher concentrations of ethanol can lead to irregular structures in the overlay agar and thus interfere with the counting of the colonies.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dihexyl fumarate

EC Number:

242-833-4

EC Name:

Dihexyl fumarate

Cas Number:

19139-31-2

Molecular formula:

C16H28O4

IUPAC Name:

dihexyl fumarate

Specific details on test material used for the study:

Identity: DIHEXYL FUMARATE
Batch No.: 9000559308
Aggregate State at Room Temperature:
Colour: liquid colourless
Purity: 98.3%
Stability in solvent: not indicated by the sponsor
Storage: room temperature, light protected
Expiration Date: May 03, 2006

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

The concentration range included two logarithmic decades. The following concentrations were tested:
10; 33; 100; 333; 1000; 2500, and 5000 μg/plate

Vehicle / solvent:

On the day of the experiment, the test item DIHEXYL FUMARATE was dissolved in ethanol (purity> 99 %, MERCK, D-64293 Darmstadt). The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 4-NOPD // 2-aminoanthracene, 2-AA

Details on test system and experimental conditions:

Characterisation of the Salmonella typhimurium Strains
The histidine dependent strains are derived from S. typhimurium strain LT2 through a mutation in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation (deletion of the uvrB gene) causes an inactivation of the excision repair system. The latter alteration also includes a deletion in the nitrate reductase and biotin genes. In the strains TA 98, TA 100 and TA 102 the R-factor plasmid pKM 101 carries umu DC analogous genes that are involved in error-prone repair and the ampicillin resistance marker. The strain TA 102 does not contain the uvrS--mutation and is excision repair proficient. Additionally, TA 102 contains the multicopy plasmid pAQ1 carrying the hisG428 mutation ( ochre mutation in the hisG gene ) and a tetracycline resistance gene.

In summary, the mutations of the TA strains used in this study can be described as follows:
Salmonella typhimurium
Strains // Genotype // Type of mutations indicated
TA 1537 // his C 3076; rfa-; uvrB-: // frame shift mutations
TA98 // his D 3052; rfa-; uvrB-;R-factor // frame shift mutations
TA 1535 // his G 46; rfa-; uvrS-: // base-pair substitutions
TA 102 // his G 428; rfa-; uvrB+;R-factor // base-pair substitutions
TA 100 // his G 46; rfa-; uvrB-;R-factor // base-pair substitutions

Regular checking of the properties of the strains regarding the membrane permeability, ampicillin- and tetracycline resistance as well as spontaneous mutation rates is performed in the laboratory of RCC Cytotest Cell Research according to B. Ames et al. and D. Maron and B. Ames. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.
The bacterial strains TA 1535, TA 1537 TA 98, TA 100 and TA 102 were obtained from Trinova Biochem GmbH (35394 Gießen, Germany).

Storage
The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO (MERCK, D-64293 Darmstadt) in liquid nitrogen.

Precultures
From the thawed ampoules of the strains 0.5 ml bacterial suspension was transferred into 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. A solution of 20 µl ampicillin (25 µg/ml) was added to the strains TA 98, TA 100, and TA 102. Additionally 20 µl tetracycline (2 µg/ml) was added to strain TA 102. This nutrient medium contains per litre:
8 g Merck Nutrient Broth (MERCK, D-64293 Darmstadt)
5 g NaCl (MERCK, D-64293 Darmstadt)

The bacterial cultures were incubated in a shaking water bath for 4 hours at 37° C.

Selective Agar
The plates with the minimal agar were obtained from E. Merck, D-64293 Darmstadt.

Overlay Agar
The overlay agar contains per litre:
6.0 g MERCK Agar Agar*
6.0 g NaCl*
10.5mg L-Histidine x HCl x H2O *
12.2 mg Biotin*
* (MERCK, D-64293 Darmstadt)

Sterilisations were performed at 121° C in an autoclave.

Rationale for test conditions:

Pre-Experiment for Toxicity
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre­ experiment were the same as described for the experiment I below (plate incorporation test).
Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment I, since the following criteria are met: Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

Dose Selection
In the pre-experiment the concentration range of the test item was 3 - 5000 µg/plate. The pre-experiment is reported as f experiment I since the criteria mentioned above were met. Based on the toxic effects observed in strain TA 102 seven concentrations were tested in both main experiments.

Evaluation criteria:

Evaluation of Results
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate with s9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. II, at 5000 µg/plate with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. I, at 1000 - 5000 µg/plate with S9, and Exp. II, at 2500, 5000 µg/plate with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

DISCUSSION OF RESULTS

The test item DIHEXYL FUMARATE was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.

 

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls, were tested in triplicate. The test item was tested at the following concentrations:

 

10; 33; 100; 333; 1000; 2500, and 5000 µg/plate

 

In both experiments, reduced background growth was observed with and without S9 mix at higher concentrations in all strains used.

 

Toxic effects, evident as a reduction in the number of revertants (below the factor of 0.5) were observed at the following concentrations (µg/plate):

 

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	/	/	/	5000
TA 1537	/	/	/	5000
TA98	/	/	/	/
TA 100	/	/	/	/
TA 102	/	1000 - 5000	/	2500, 5000

/ no toxic effects observed

 

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with DIHEXYL FUMARATE at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

 

Appropriate reference mutagens were used as positive controls. They showed a distinct in­ crease in induced revertant colonies.

 

In experiment II with metabolic activation, the number of colonies did not quite reach the lower limit of our historical control data in the negative control of strain TA 1535. Since this deviation is rather small, this effect is judged to be based upon biological fluctuations and has no detrimental impact on the outcome of the study.

 

The historical range of positive controls was exceeded in strains 1537 and TA 98 (experiment II) with metabolic activation. This effect indicates the sensitivity of the strains rather than compromising the assay.

 

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Applicant's summary and conclusion

Conclusions:

During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.